Pharmaceutical composition for treating or alleviating autoimmune-related diseases and method for treating or alleviating autoimmune-related diseases

ABSTRACT

A pharmaceutical composition for treating or alleviating autoimmune-related diseases is provided, including an extract of  Amomum tsao - ko  as an active ingredient and a pharmaceutically acceptable carrier, in which the extract comprises Vanillin, Tsaokoin, or the combination thereof.

CROSS REFERENCE TO RELATED APPLICATION

The present application claims the priority benefits of U.S. provisionalapplication Ser. No. 62/287,345, filed on Jan. 26, 2016. The entirety ofthe above-mentioned patent application is hereby incorporated byreference herein and made a part of this specification.

TECHNICAL FIELD

The technical field relates to pharmaceutical compositions for treatingor alleviating autoimmune-related diseases.

BACKGROUND

Under a normal physiological condition, immune system can be regarded asthe defense army of the body. When immune system detects the invasion ofbacteria, virus, or other foreign materials, it will produce antibodiesand attack these foreign materials. However, when immune system isdisorder and no longer be able to accurately distinguish between theenemy and the self, the defense army will attack their own tissues ororgans, and trigger the so-called “Autoimmune disease”.

The prevalence of suffering from autoimmune disease is about 5% of theglobal population. The type of autoimmune disease ranges from singleorgan disease (such as Hashimoto's thyroiditis) to systemic diseases(such as systemic lupus erythematosus, referred to SLE). Thepathological features of autoimmune disease include that a large numberof abnormal infiltration lymphocytes can be found in the inflammatorytissues or organs, and the autoantibody can be detected in the blood ofthe patient.

In addition, the specific autoimmune diseases are regionally distributedand may be related to race, gene and environment. In the case ofmultiple sclerosis (MS), it usually occurs in the ancestors of theCaucasian race from Northern Europe, and the residents of high latitudesare more likely to suffer from MS. The current study shows that MS is achronic inflammatory disease caused by auto-demyelination in centralnerve system, and due to the damaged nerve is different, the clinicallesions of individual patient are different, including loss of balance,spasm, difficulty in hands and feet, paralysis, blurred vision and othersymptoms.

Regarding the pathogenic mechanism of autoimmune diseases, many studieshave pointed that overexpression of interferon or tumor necrosis factormay result in rheumatoid arthritis, Crohn's disease, psoriasisarthritis, and other immune-related diseases. Recent studies have shownthat interleukin, such as IL-17, regulated by T helper cells, is also animportant cytokine for dominating autoimmune reaction.

In the past, the main drugs used to treat autoimmune diseases includenon-steroidal anti-inflammatory drugs, steroids, immune-modulators, andimmune-suppressive agents. However, these drugs are known to have therisk of side effects, and somehow they may interact with each other.

In view of this, the new generation of therapeutic drugs are developedto adopt immuno-target therapy, which designs biological agentstargeting for specific immune messages or molecules in autoimmunediseases, and these biological agents can accurately target abnormalimmune molecules, but will not hurt the normal cells.

Therefore, there is an urgent demand to develop a low side-effect andspecific biological agents at present.

SUMMARY

The disclosure provides a pharmaceutical composition for treating oralleviating autoimmune-related diseases, comprising: an extract ofAmomum tsao-ko as an effective ingredient and a pharmaceuticallyacceptable carrier or salt.

The disclosure also provides a pharmaceutical composition for treatingor alleviating autoimmune-related diseases, comprising: a compound as aneffective ingredient and a pharmaceutically acceptable carrier or salt,wherein the compound is vanillin, tsaokoin, or a combination thereof.

The disclosure also provides a method of treating or alleviatingautoimmune-related diseases, comprising: administering a pharmaceuticalcomposition to a subject in need thereof, wherein the pharmaceuticalcomposition comprises an extract of Amomum tsao-ko as an effectiveingredient and a pharmaceutically acceptable carrier or salt.

Furthermore, the disclosure provides a method of treating or alleviatingautoimmune-related diseases, comprising: administering a pharmaceuticalcomposition to a subject in need thereof, wherein the pharmaceuticalcomposition comprises a compound as an effective ingredient and apharmaceutically acceptable carrier or salt, wherein the compound isvanillin, tsaokoin, or a combination thereof.

The present disclosure can be more fully understood by reading thesubsequent detailed description and exemplary embodiments withreferences to the accompanying drawings so as to be easily realized by aperson having ordinary knowledge in the art, wherein:

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1 shows the effect of Amomum tsao-ko extract HE-06 on TNF-αsecretion in LPS-induced acute inflammatory BALB/c mouse model. **represents p value is less than 0.01;

FIG. 2A shows that Amomum tsao-ko extract HE-06 exhibits the effect ondelaying the progression of experimental autoimmune encephalomyelitis(EAE) in C57BL/6 mouse model, in which the results were obtained throughdaily observation and recording for 14 consecutive days, and the scoringcriteria of the progression is based on Ataxia score and EAE score whichhave been recognized in literature. * represents p value is less than0.05; ** represents p value is less than 0.01; *** represents p value isless than 0.001;

FIG. 2B-2C show the effects of the extracts, 2015-0803-2 and2015-0909-2, on the progression of EAE mouse model, respectively. Inwhich 2015-0803-2 and 2015-0909-2 are the further extracts of Amomumtsao-ko extract HE-06 and obtained through the toxicity-attenuatingsteps, and both 2015-0803-2 and 2015-0909-2 extracts are administeredwith a dose of 250 mg/kg. In which ** represents p value is less than0.01; *** represents p value is less than 0.001;

FIG. 3A-3E show the effects of active fractional interval (I) of Amomumtsao-ko extract HE-06 on EAE acute inflammatory mouse model, includingdelaying the development of EAE progression, improving EAE disorders,improving demyelination of the cervical spine, and reducing theincidence of demyelination and axonal swelling in the entire spinalcord, respectively. In which * represents p value is less than 0.05; **represents p value is less than 0.01; *** represents p value is lessthan 0.001;

FIG. 4A-4C show the effects of active fractional interval (II) of Amomumtsao-ko extract HE-06 on inhibition of TNF-α secretion in LPS-inducedacute inflammatory mouse model, delaying the progression of EAE mousemodel, and slowing down the skin inflammatory conditions of IMQ-inducedpsoriasis-like dermatitis, respectively. In which * represents p valueis less than 0.05; ** represents p value is less than 0.01; ***represents p value is less than 0.001;

FIG. 5A-5D show the effects of active ingredient Tsaokoin (TK) of Amomumtsao-ko extract HE-06 on inhibiting IL-17 secretion of induced EL4lymphoma cells, inhibiting TNF-α secretion in LPS-induced acuteinflammatory mouse model, delaying the progression of EAE mouse model,and slowing down the skin inflammatory conditions of IMQ-inducedpsoriasis-like dermatitis, respectively. In which * represents p valueis less than 0.05; ** represents p value is less than 0.01; ***represents p value is less than 0.001.

DETAILED DESCRIPTION

In the following detailed description, for purposes of explanation,numerous specific details are set forth in order to provide a thoroughunderstanding of the disclosed embodiments. It will be apparent,however, that one or more embodiments may be practiced without thesespecific details. In other instances, well-known structures and devicesare schematically shown in order to simplify the drawing.

The disclosure provides a pharmaceutical composition for treating oralleviating autoimmune-related diseases, which comprises an effectiveamount of an extract of Amomum tsao-ko (hereinafter referred to as A.tsao-ko) as a main effective ingredient and a pharmaceuticallyacceptable carrier or salt. The above-mentioned A. tsao-ko is belong toevergreen perennial herb of Zingiberaceae cardamom genus, with a heightof 2.5 to 3 meters and grown in forest humid zone of tropical andsubtropical. Otherwise, the origin of A. tsao-ko is distributed inYunnan of China and Southeast Asia, and its artificial cultivationhistory has been 200 years. In addition to herbal medicine, the dryfruit of A. tsao-ko can also be used as seasonings because of its wholeplant having spicy flavor. Hence, A. tsao-ko can be considered as bothfood and medicine material.

The above-mentioned extract of A. tsao-ko is extracted from the root,stem, leaf, flower, fruit, seed, bark or a combination thereof. In oneembodiment, the above-mentioned extract of A. tsao-ko can be extractedfrom the fruit of A. tsao-ko.

The above-mentioned extract of A. tsao-ko is obtained from performing anextraction with an organic solvent. The said organic solvent maycomprise alcohol, ester, alkane, haloalkane, or a combination thereof,but it is not limited thereto.

The above-mentioned alcohol may be C1-C12 alcohols (for example,methanol, ethanol, propanol, isopropanol, n-butanol, 2-butanol,t-butanol, 1,3-butanediol, 1,4-butanediol, pentanol, isoamyl alcohol,2,3-pentanediol, 2,4-pentanediol, cyclopentanol, hexanol, cyclohexanol,heptanol, octanol, nonanol, decanol, hendecanol, dodecanol, etc.), theabove-mentioned esters may be C2-C5 acid esters (for example, ethylacetate, propyl acetate, amyl acetate, amyl propionate, etc.), theabove-mentioned alkanes may be C5-C6 alkanes (for example, n-heptane,n-pentane, cyclopentane, n-hexane, cyclohexane, etc.), and theabove-mentioned haloalkane may be, for example, methyl chloride or ethylchloride, but it is not limited thereto.

In one embodiment, the above-mentioned extract of A. tsao-ko can beobtained from performing an extraction with alcohol solvent. In anotherembodiment, the extract of A. tsao-ko can be obtained from performing anextraction by alcohol solvent with the concentration of ranging from 50to 95%. In which, the concentration of the alcohol solvent utilized toperform the extraction of A. tsao-ko can be 50-60%, 60-70%, 70-80%,80-90%, or 90-95%. In another embodiment, the concentration of thealcohol solvent utilized to perform the extraction of A. tsao-ko can be95% alcohol solvent.

Before extraction, the weight of A. tsao-ko which would be used thistime is calculated first, and the extraction is then carried out by theproportion of which a volume of the organic solvent is 3-10 times theweight of A. tsao-ko. In one embodiment, the volume of the organicsolvent can be 3-7 times the weight of A. tsao-ko. In anotherembodiment, the volume of the organic solvent can be 5 times the weightof A. tsao-ko. In addition, the extraction is performed an extractiontemperature of 10 to 35° C. and an extraction time of 3 to 10 days. Inone embodiment, the extraction temperature can be 20 to 30° C. and theextraction time can be 5 to 8 days. In another embodiment, theextraction temperature can be 25° C. and the extraction time can be 7days.

In one embodiment, the extraction of the extract of A. tsao-ko isfurther performed with a shaking mode, including horizontal shaking,vertical shaking, rotate shaking, 3D-nutating shaking, seesaw shaking,ultrasonic shaking or a combination thereof, but it is not limitedthereto. The extract of A. tsao-ko obtained by the preceding extractionprocess is still passed through a suction filtration step and theresulting filtrate is considered as the test substance of the extract ofA. tsao-ko, hereinafter referred to as HE-06.

In one embodiment, the extraction of the above-mentioned extract of A.tsao-ko HE-06 further comprises a distillation step. In anotherembodiment, the extraction of the above-mentioned extract of A. tsao-koHE-06 still comprises a heating reflux step with a low polarity solvent.

Otherwise, in one embodiment, the pharmaceutical composition comprisingthe above-mentioned extract of A. tsao-ko HE-06 as an effectiveingredient has the effect on inhibiting cytokine secretion, and the saidcytokine includes IL-17, TNF-α, and a combination thereof, but it is notlimited thereto.

In another embodiment, the pharmaceutical composition comprising theabove-mentioned extract of A. tsao-ko HE-06 as an active ingredient canbe used for treating or alleviating autoimmune-related diseases. Thesaid treating or alleviating autoimmune-related diseases can include,but is not limited to, alleviating the syndrome of theautoimmune-related diseases or slowing down the progress of theautoimmune-related diseases. In which, the said autoimmune-relateddiseases can include Multiple Sclerosis (MS), neuromyelitis optica(NMO), acute disseminated encephalomyelitis (ADEM),demyelination-related neuritis, Ankylosing spondylitis, Psoriasis,Psoriatic arthritis, Rheumatoid arthritis (RA), Crohn's disease,Juvenile idiopathic arthritis (JIA), Ulcerative Colitis (UC), and acombination thereof, but it is not limited thereto. In one embodiment,the pharmaceutical composition comprising the above-mentioned extract ofA. tsao-ko HE-06 as an effective ingredient can be used for treating oralleviating Multiple Sclerosis.

In another embodiment, the pharmaceutical composition comprising theabove-mentioned extract of A. tsao-ko HE-06 as an effective ingredientmay be administered orally, non-orally, parenterally by an inhalationspray, or via an implanted reservoir. The parenteral method may comprisesubcutaneous, intracutaneous, intravenous, intramuscular,intra-articular, intra-arterial, intrasynovial, intrasternal,intrathecal, and intralesional injection or infusion techniques, but itis not limited thereto.

An oral composition can comprise, but is not limited to, tablets,capsules, emulsions, aqueous suspensions, dispersions and solutions.

In one embodiment, the pharmaceutical composition can be administered byoral or subcutaneous injection. It is also possible to administer withmulti-dose thereof in a suitable period according to the pharmacologicalroutine method for determining the course of administration to which thepatient is applied.

The pharmaceutical composition comprising the above-mentioned extract ofA. tsao-ko HE-06 as an effective ingredient further comprises apharmaceutically acceptable carrier or salt well known in the art andadded in appropriate proportion. The pharmaceutically acceptable carriermay include, but is not limited to, a solvent, a dispersion medium, acoating, an antibacterial and antifungal agent, or an isotonic andabsorption delaying agent, etc. which is compatible to pharmaceuticaladministration. The pharmaceutical composition can be formulated intodosage forms for different administration routes utilizing conventionalmethods.

Moreover, the pharmaceutically acceptable salt may include, but is notlimited to, inorganic cation salt, such as alkali metal salts such assodium salt, potassium salt or amine salt, such as alkaline-earth metalsalt such as magnesium salt or calcium salt, such as the salt containingbivalent or quadrivalent cation such as zinc salt, aluminum salt orzirconium salt. In addition, the pharmaceutically acceptable salt mayalso comprise organic salt, such as dicyclohexylamine salt,methyl-D-glucamine, and amino acid salt such as arginine, lysine,histidine, or glutamine.

In one embodiment of the present disclosure, a method of treating oralleviating autoimmune-related diseases is also provided, in which themethod comprises administering a pharmaceutical composition to a subjectin need thereof, and the pharmaceutical composition comprises an extractof A. tsao-ko as an effective ingredient and a pharmaceuticallyacceptable carrier or salt. The said subject can include mammals, suchas rat, dog, cat, horse, sheep, pig, monkey, ape, etc., especiallyhuman.

In another embodiment of the present disclosure, a pharmaceuticalcomposition for treating or alleviating autoimmune-related diseases isprovided, in which the pharmaceutical composition comprises a compoundas an effective ingredient and a pharmaceutically acceptable carrier orsalt, and the compound is vanillin, tsaokoin, or a combination thereof.

In which, the above-mentioned vanillin and tsaokoin are derived from theextract of A. tsao-ko, HE-06, which is obtained by performing theextraction with the proportion of organic solvent volume/A. tsao-koweight=3-10/1, at 10-35° C. for 3 to 10 days shaking.

In one embodiment, the above-mentioned extract of A. tsao-ko, HE-06, isfurther extracted with a distillation step to remove the portion ofessential oil. Then, a step of heating and refluxing with a low polaritysolvent is performed to reduce the toxicity and extract the activeingredient therein. The said low polarity solvent can include petroleumether, C4-C9 linear alkanes (such as n-Hexane), n-Heptane, C4-C9cycloalkanes, unsaturated benzene, esters, ketones, and a combinationthereof, but it is not limited thereto.

With regard to the extraction of the extract of A. tsao-ko, includingthe above-mentioned extraction site, the type, concentration and volumeof the utilized organic solvent, the extraction condition employed inthe extraction process including the extraction temperature, time,accompanied shaking mode, etc., as described in the above description,will not repeat them here.

In another embodiment, the pharmaceutical composition comprisingvanillin, tsaokoin or a combination thereof as an effective ingredienthas the effect on inhibiting cytokine secretion, and the cytokineincludes IL-17, TNF-α, and a combination thereof, but it is not limitedthereto.

In yet another embodiment, the pharmaceutical composition comprisingvanillin, tsaokoin or a combination thereof as an effective ingredientcan be can be used for treating or alleviating autoimmune-relateddiseases. The said treating or alleviating autoimmune-related diseasescan include, but is not limited to, alleviating autoimmune-relateddiseases or slowing down autoimmune-related diseases. In which, the saidautoimmune-related diseases can include Multiple Sclerosis (MS),neuromyelitis optica (NMO), acute disseminated encephalomyelitis (ADEM),demyelination-related neuritis, Ankylosing spondylitis, Psoriasis,Psoriatic arthritis, Rheumatoid arthritis (RA), Crohn's disease,Juvenile idiopathic arthritis (JIA), Ulcerative Colitis (UC), and acombination thereof, but it is not limited thereto. In one embodiment,the pharmaceutical composition comprising vanillin, tsaokoin or acombination thereof as an effective ingredient can be used for treatingor alleviating Multiple Sclerosis.

In another embodiment, the pharmaceutical composition comprisingvanillin, tsaokoin or a combination thereof as an effective ingredientmay be administered orally, non-orally, parenterally by an inhalationspray, or via an implanted reservoir. With regard to the form of oral ornon-oral administration, and the medication administration, as describedin the above description, will not repeat them here.

In addition, the pharmaceutical composition comprising vanillin,tsaokoin or a combination thereof as an effective ingredient furthercomprises a pharmaceutically acceptable carrier or salt well known inthe art and added in appropriate proportion. With regard to the speciesof pharmaceutically acceptable carrier or salt, as described in theabove description, will not repeat them here.

Hence, in another embodiment of the present disclosure, a method oftreating or alleviating autoimmune-related diseases is provided, inwhich the method comprises administering a pharmaceutical composition toa subject in need thereof, wherein the pharmaceutical compositioncomprises a compound as an effective ingredient and a pharmaceuticallyacceptable carrier or salt, wherein the compound is vanillin, tsaokoin,or a combination thereof. With regard to the subject to which isadministered, as described in the above description, will not repeatthem here.

Examples

The specific embodiments of the present disclosure are described asfollows. However, the following examples are merely illustrative of thetechnical aspects of the present invention and should not be construedas limiting the scope of the invention.

Example 1: Preparation of the Extract of A. tsao-Ko HE-06

A. Material

The fruit of A. tsao-ko was taken as the medicinal material. Aftercleaning and drying, the fruit of A. tsao-ko was initially crushed byherbs crusher.

B. Method

About 500 g of the crude crushed of A. tsao-ko was immersed in 5-foldvolume (about 2500 mL) of 95% ethanol at 25° C. with shaking of 125 rpmin shaker for 7 days. The extract was then collected for suctionfiltration. Thereafter, the filtered filtrate was quantified and sampled2 mL for analysis by high performance liquid chromatography (HPLC) andthin film chromatography (TLC). The remaining filtrate was concentratedunder reduced pressure to a volume of about 30 mL, which was thendispensed and freeze-dried to be the test substance of the extract of A.tsao-ko, hereinafter referred to as HE-06.

Example 2: The Extract of A. tsao-Ko HE-06 can Inhibit IL-17 Secretion

A. Anti-IL-17 Secretion in EL4 (Murine Lymphoma) Cells (1) Cell Culture

EL4 cell line is murine lymphoma cell purchased from BioresourceCollection and Research Center (referred to as BCRC) and cultured inRPMI 1640 medium which contains 10% Fetal Bovine Serum (referred to asFBS), 1.5 g Sodium bicarbonate, 1 mM Sodium pyruvate, 1× non-essentialamino acids (referred to as NEAA), and 0.5 mM 2-Mecaptoethanol (referredto as 2-ME). The subculture was performed 2-3 times a week and theculture condition was according to the suggestion of BCRC.

(2) IL-17 Secretion and MTT Test

EL4 lymphoma cells were seeded into 96-well plate by 1×10⁵ cells perwell and 10 μl of the extract of A. tsao-ko HE-06 was added to each welland incubated at 37° C. in 5% CO₂ cell incubator for 1 hour. Then,Phorbol-12-myristate-13-acetate (referred to as PMA or TPA) andIonomycin were added to be the stimulants that induce activation oflymphoma cells, in which the final concentration of PMA was 10 ng/ml andthe final concentration of Ionomycin was 5 ng/ml, and additional 18hours incubation was executed. In the above test, the group of addingboth PMA (final concentration of 10 ng/ml) and Ionomycin (finalconcentration of 5 ng/ml) was considered as the control group.

Thereafter, the cell-containing 96-well plate was centrifuged at 1200rpm for 5 minutes and the supernatant was collected and transferred tothe new 96-well plate. 5 μl MTT of 5 mg/ml was added to the cells inoriginal 96-well plate and 1 hour incubation of 37° C. and 5% CO₂ incell incubator was executed to generate crystal violet. 150 μl DMSO wasthen added to dissolve the crystal violet and the absorbance of 570 nmwas measured to assess the cell viability.

As for the supernatant in new 96-well plate, mouse IL-17 ELISA kit (R&D,DY421E) was used to detect the IL-17 secretion, and the foregoing IL-17amount in supernatant represents the IL-17 amount secreted by the cells.In addition, the detection results of IL-17 secretion were firstnormalized by the control group as 100%, and IC₅₀ of the extract of A.tsao-ko HE-06 on inhibiting IL-17 secretion was calculated. The resultswere shown in Table 1.

B. Anti-IL-17 Secretion in Rat PBL (Peripheral Blood Leukocyte) (1) CellCulture

The blood of SD rats (Sprague Dawley rat) was collected by means ofsapheneous vein sampling and then placed in EDTA-containing centrifugetubes (the final concentration of EDTA was 2 mg/ml) as well as mixedhomogeneously. Then about 4-fold the total volume of blood and EDTA ofred blood cell lysate (ACK Lysis Buffer or Red Blood Cell Lysis Buffer)was added to break the red blood cells. The reaction was carried out at37° C. water bath for 5 minutes, and then about 4-fold the total volumeof blood, EDTA and erythrocyte lysate of buffer (PBS buffer) was addedand the supernatant was removed by centrifugation at 3000 rpm for 5minutes. The foregoing step of breaking the red blood cells was repeatedtwice, and then centrifugation was performed to obtain the precipitatedpart, namely PBL. Thereafter the appropriate volume of RPMI cell culturemedium was added for culture.

(2) Test of Inhibition in IL-17 Secretion

PBL cells were seeded into 96-well plate by 2×10⁵ cells per well and 10μl of the extract of A. tsao-ko HE-06 was added to each well andincubated in 37° C. and 5% CO₂ cell incubator for 1 hour. Then, (finalconcentration of 10 ng/ml) and Ionomycin (final concentration of 5ng/ml) were added to be the stimulants that induce activation oflymphoma cells, and additional 48 hours incubation was executed. In theabove test, the group of adding both PMA (final concentration of 10ng/ml) and Ionomycin (final concentration of 5 ng/ml) was considered asthe control group.

Thereafter, the cell-containing 96-well plate was centrifuged at 3000rpm for 5 minutes and the supernatant was collected and transferred tothe new 96-well plate. 5 μl AlamarBlue was added to the cells inoriginal 96-well plate and additional 4 hours incubation of 37° C. and5% CO₂ in cell incubator was executed. Then, the cell viability wasassessed by ELISA reader to measure the absorbance of excitation lightat 560 nm as well as the emitting light at 590 nm.

As for the supernatant in new 96-well plate, rat IL-17 ELISA kit(eBioscience, 88-7170) was used to detect the IL-17 secretion. Inaddition, the detection results of IL-17 secretion were first normalizedby the control group as 100%, and IC₅₀ of the extract of A. tsao-koHE-06 on inhibiting IL-17 secretion was calculated. The results werealso shown in Table 1.

TABLE 1 The inhibitory effects of HE-06 on IL-17 secretion in EL4 murinelymphoma cells and rat Peripheral Blood Leukocytes. IC₅₀ is the HE-06concentration causing 50% IL-17 secretion inhibition. IC₅₀ (μg/ml) Testsample EL4 PBL HE-06 27.9 9.9

The results of in vitro cell analysis shown in Table 1 indicated thatthe extract of A. tsao-ko HE-06 can inhibit the secretion of IL-17 inPMA and Ionomycin-induced EL4 lymphoma cells and peripheral bloodleukocytes.

Example 3: The Extract of A. tsao-Ko HE-06 can Inhibit TNF-α Secretion

(1) Experimental Animal

The 6-8 weeks BALB/c male mice were purchased from BioLASCO Taiwan Co.,Ltd. In addition to make the feeding condition meeting with nationalexperimental animal guidelines, the batch of mice were marked, sub-cageand weighing soon after acquisition. Then, these mice were placed to thequarantine room of general area for a week of quarantine work. Duringthe quarantine period, observation of mobility and environmentaladaptability was performed, and after assessment to be normal, thesemice were transferred into the feeding area for experimentalimplementation.

The light of feeding area was auto-controlled by 12 hours light and 12hours dark, as well as the room temperature was controlled at 23±2° C.During feeding, mice were free to obtain adequate food and drinkingwater. Due to abundant basic references and related data have beenestablished for this laboratory animal strain, it is applicable toinflammatory functional assessment test. The experimental methodsestablished in the following inflammatory models have been approved bythe IACUC Committee of ITRI.

(2) Establish the Model of Acute Inflammation in Mice

Before the experiment, the mice were weighed and grouped so that theaverage body weight of each group was not significantly different. Thenthe mice were fasted for 2 to 4 hours but regular drinking water wasstill supplied. LPS stimulation, and the control group was given thesame volume of solvent. After administration, the clinical symptoms ofthe mice were observed and recorded. Thereafter, LPS stimulation (about0.25 ml/mouse) was conducted by IP injection. In which, the mice inexperimental group were administered orally with the extract of A.tsao-ko HE-06 2 hours before LPS stimulation, and the mice in controlgroup were administered with the same volume of solvent. Afteradministration, the clinical symptoms of the mice were observed andrecorded.

After 1.5 hours LPS stimulation, the mice were euthanized by excessiveCO₂ and the whole blood was collected by cardiac puncture bloodsampling. Next, the plasma was collected after centrifugation at 6000rpm and 4° C. for 10 minutes and then TNF-α secretion were analyzed.

(3) Detection and Analysis of TNF-α Secretion

The TNF-α secretion in plasma was analyzed according to the followingprocedure shown in Duoset® ELISA kit. First, 100 μl of the CaptureAntibody mixture solution was added to each well of the 96-well plate atroom temperature to cover the surface of each well overnight. Afterthat, the mixture solution was dried and washed with 300 μl Wash Bufferper well three times. Next, 200 μl of Block buffer was added at roomtemperature for 1 hour, and the previous wash step was repeated.Thereafter, 100 μl test sample (the foregoing plasma) and standard ofappropriate dilution were added to each well to stand at roomtemperature for 2 hours, and the aforementioned washing step wasrepeated. Then, 100 μl of Detection Antibody mixture solution was addedto each well to stand at room temperature for 2 hours, and theaforementioned washing step was repeated again. Thereafter, 100 μl ofStreptavidin-HRP mixture solution was added to each well for reaction atroom temperature away from light for 20 minutes. After repeating theaforementioned washing step, 100 μl of Substrate Solution (TMB) wasadded to each well for reaction at room temperature away from light for20 minutes. Finally, 50 μl of Stop Solution (1N HCl) was added to eachwell to stop the color reaction, and the absorbance of OD₄₅₀ nm wasmeasured.

The experimental results were expressed by mean±standard error (referredto as S.E.), and t-test statistics was used to distinguish thedifferences between the groups. ** represents that p value of less than0.01 and indicates that there exists a statistically significantdifference between the two groups. The experimental results are shown inFIG. 1.

As shown in FIG. 1, the extract of A. tsao-ko HE-06 could inhibit thesecretion of TNF-α in LPS-induced acute inflammatory mice, and theinhibition level was up to 34%, indicating that the extract of A.tsao-ko HE-06 could effectively in vivo inhibit TNF-α secretion inducedby inflammation.

Example 4: The Extract of A. tsao-Ko HE-06 can Slow Down the Progress ofExperimental Autoimmune Encephalomyelitis (EAE) and Improve the ClinicalSymptoms of EAE

(1) Experimental Animal

The experimental animals, female C57BL/6 mice, aged about 8 weeks, werepurchased from National Laboratory Animal Center (Tainan). Afteracclimation and quarantine, the mice were approximately 10-12 weeks ofage at the time of performing the EAE experiments. The experimentalanimals were bred in the environment: 12 hours light and 12 hours dark,room temperature of 23±2° C., and relative humidity of 40-70%. Duringfeeding, animals were free to obtain adequate food and drinking water,but after the onset of EAE, the soaked soft feed and agar gel jelly wereespecially given in the bottom of the feeding cage, so that the onsetanimals were easier to feed and drink. In addition, during thequarantine and testing period, the animals were observed and recorded bythe veterinarian and the experimental staff of ITRI to ensure health ofthese experimental animals.

(2) Establishment of Autoimmune Mouse Model of EAE

The foregoing C57BL/6 mice were administered subcutaneously with 200 μlemulsion including 200 μg MOG35-55 peptide and 400 μg Mycobacteriumtuberculosis H37Ra, followed by intraperitoneal injection of 500 ngpertussis toxin for induction of EAE. From the 7^(th) day afterinduction, the EAE clinical symptoms were recorded daily according tothe recognized Ataxia score (grading of nervous system disorder) and EAEscore as the scoring criteria (description of Ataxia score and EAE scoreare as follows). When the experimental animals began to appear Ataxiascore 0++, followed by S-type grouping, and began to administrate theextract of A. tsao-ko HE-06 for 14 days. During the period, thecondition of the mice was observed daily and their clinical symptomswere recorded according to the EAE score, and the group onlyadministered with solvent was considered as the control group.Thereafter, animals were sacrificed on the 15^(th) day, and blood orrelated organs were collected for subsequent analysis.

(3) Records and Data Analysis of Ataxia Score and EAE Score

The criteria of Ataxia score are as follows: 0+ represents that the rearfoot of the animal is splayed while moving, swing left and right, andgait imbalance; 0++ represents that when grasping the rear neck of theanimal to observe whether its tail can be raised by itself and to testthe tensile strength of its tail by fingers, the tail appears unable tobe raised by itself as well as reduced tension.

The criteria of EAE score are as follows: Score 0 represents no EAEsymptoms; Score 0.5 represents temporal weak tail, sometimes raised;Score 1 represents limp tail, unable to lift normally; Score 2represents paralyzed tail or slightly hind limb weakness; Score 3represents moderate to severe hind limb paralysis or mild forelimbweakness; Score 4 represents complete hind limb paralysis or moderate tosevere forelimb weakness; Score 5 represents limb paralysis accompaniedby incontinence or presenting a dying state; Score 6 represents death.

The experimental results were expressed by mean±standard error of mean(referred to as S.E.M.), and t-test statistics was used to distinguishthe differences between the groups. In which * represents p value ofless than 0.05; ** represents p value of less than 0.01; *** representsp value of less than 0.001 and these asterisk indicate that there existsa statistically significant difference between the two groups. Theexperimental results are shown in FIG. 2A.

As shown in FIG. 2, after induction of EAE, the mice have been to showthe clinical symptoms of hind limb paralysis and severe forelimbweakness (such as Score 4 described above) on 14^(th) day after onset,but administering the extract of A. tsao-ko HE-06 could significantlyslow down the progress of EAE, indicating that the extract of A. tsao-koHE-06 could effectively improve the clinical symptoms in EAE animalmodel.

Example 5: Purification, Preparation and Toxicity-Attenuation Test ofthe Active Fractional Interval (I) of HE-06

The extract of A. tsao-ko, HE-06 was further extracted with adistillation step by steam for 24 hours to remove the portion ofessential oil. Then, a step of heating and refluxing with a specificsolvent was performed for 8 hours to remove the dregs and reduce thetoxic portion.

With regard to the step of reducing the toxic portion, if the solvent{n-Hexane:Ethyl acetate=1:1} was utilized to perform the step of heatingand refluxing for 8 hours, the resulting extract was designated as2015-0803-2. Further, by EAE mouse model test, the results indicatedthat although the clinical symptoms of the ten EAE mice administeredwith 2015-0803-2 of 250 mg/ml have been improved (as shown in FIG. 2B),still three mice were resulted to death.

However, if the solvent was replaced with the low polarity solventn-Hexane to perform the step of heating and refluxing for 8 hours, andthen the extract was eluted by gradient elution with n-Hexane, EtOAc andMeOH (i.e., the solvent with lower polarity is utilized to separate thematerial which is easily eluted, and then the amount of solvent withhigher polarity is gradually increased, so that the closely adsorbedmaterial can be removed from the column), and the step of Silica GelColumn Chromatography was further performed. Each fractional intervalwas assessed via the cell activity test and the optimal activefractional interval was obtained, in which the extract was designated as2015-0909-2. In the Same way, by EAE mouse model test, the resultsindicated that the clinical symptoms of the ten EAE mice administeredwith 2015-0909-2 of 250 mg/ml have been improved (as shown in FIG. 2C)and none mice were resulted to death. In which * represents p value ofless than 0.05; ** represents p value of less than 0.01; *** representsp value of less than 0.001.

Hence, the extract 2015-0909-2 with the better toxicity-attenuatingeffect was considered as the active fractional interval (I) of HE-06.

Example 6: The Active Fractional Interval (I) of HE-06 can Improve theClinical Symptoms of EAE

Except for the drugs to be administered was replaced with the activefractional interval (I) of HE-06 and the administered active fractionalinterval (I) of HE-06 with the concentration of 0 mg/ml (as the controlgroup or vehicle), 30 mg/ml, and 100 mg/ml, the experimental animals,the established of mouse autoimmune EAE model, and the scoring criteriaare the same as described above, and will not repeat them here. Theexperimental results were shown in FIG. 3A-3E. In which the groupadministered with solvent of the equal volume was considered as thecontrol group, and the group administered with 5 mg/mlmethylprednisolone (a steroid medication, can be used foranti-inflammation) was considered as the positive control group.

As shown in FIGS. 3A and 3B, after induction of EAE, the mice have beenappeared the clinical symptoms of hind limb paralysis and severeforelimb weakness (such as Score 4 described above) on 14^(th) day afteronset, but administration of the active fractional interval (I) of HE-06with the concentration of 30 mg/ml and 100 mg/ml could significantlyslow down the progress of EAE. In which * represents p value of lessthan 0.05; ** represents p value of less than 0.01; *** represents pvalue of less than 0.001.

Otherwise, Luxol fast blue (LFB) stain was performed to check the myelinof nerve tissue. Since myelin is a tubular sheath that is wrappedoutside the axons of the neuronal cell, the integrity of the myelinunder normal or pathological conditions can be observed by performingthe myelin staining. As the histopathology staining pattern shown inFIG. 3C, the active fractional interval (I) of HE-06 could significantlyimprove the symptom of demyelination of cervical vertebrae in EAE animalmodel.

Moreover, the condition of the spinal cord was checked with theguidelines of Histopathological Lesion Severity Scores in the literaturepublished by Shackelford et al. The histopathological grade and thedegree of myelination damage of the spinal cord including cervicalvertebrae, thoracic vertebrae and lumbar vertebrae were evaluated by apathologic veterinarian. In which, the grade of histopathology isdivided into five grades: Grade 1 represents the smallest damage(overall damage area<1%); Grade 2 represents minor damage (approximately1-25% of total damage area); Grade 3 represents moderate Grade 4represents moderate to severe injury (approximately 51-75% of totaldamage area); Grade 5 represents severe injury (approximately 76-100% oftotal damage area).

The observed and scored results were shown as FIGS. 3D and 3E. Afterinduction of EAE, the mice have been to show the clinical symptoms ofdemyelination of spinal cord and axonal swelling on 14^(th) day afteronset, but administering the active fractional interval (I) of HE-06with the concentration of 30 mg/ml and 100 mg/ml could significantlyreduce the degree of damage of the spinal cord in EAE animal model. Inwhich * represents p value of less than 0.05; ** represents p value ofless than 0.01.

Example 7: Purification and Preparation of the Active FractionalInterval (II) of HE-06

2.19 kg of HE-06 was taken to perform the extraction with 16 L methanolby heating and refluxing for 2 hours, and the extraction step wasrepeated twice to remove the dregs. The two extracts were then combinedand concentrated to 100 mL, and 1900 mL of water was added to uniformlydisperse the extract. Next, the extract was extracted with n-heptane andethyl acetate twice, respectively, the ethyl acetate extract wasconcentrated to concrete type of 26.07 g, and 21.27 g of the ethylacetate concrete was extracted by performing the solid phase adsorptionextraction. Thereafter, 900 mL ethyl acetate solution with 80%n-heptane, 1800 mL ethyl acetate solution with 60% n-heptane, 3600 mLethyl acetate solution with 55% n-heptane, 900 mL ethyl acetate solutionwith 40% n-heptane, and 900 mL ethyl acetate solution were sequentiallyadded to perform the elution. Every 900 mL was collected in a bottle andnumbered No. 1˜9, in which No. 3 was the active fractional interval (II)of HE-06.

Example 8: The Active Fractional Interval (II) of HE-06 can InhibitIL-17 and TNF-α Secretion

(1) Test of Inhibition in IL-17 Secretion

Except for the drugs to be administered was replaced with the activefractional interval (II) of HE-06, the utilized EL4 lymphoma cells, theculture condition, and the detection method of IL-17 secretion are thesame as described above, and will not repeat them here. The detectedresult of IL-17 secretion was shown in Table 2.

TABLE 2 The inhibitory effect of the active fractional interval (II) ofHE-06 on IL-17 secretion in EL4 murine lymphoma cells. IC₅₀ is theconcentration causing 50% IL-17 secretion inhibition. Test sample IC₅₀(μg/ml) active fractional interval (II) of HE-06 17.4

The results of in vitro cell analysis shown in Table 2 indicated thatthe active fractional interval (II) of HE-06 can inhibit the secretionof IL-17 in activated EL4 lymphoma cells.

(2) Test of Inhibition in TNF-α Secretion

Except for the drugs to be administered was replaced with the activefractional interval (II) of HE-06, the utilized BALB/c mice, the feedingcondition, the mouse model of acute inflammation, and the detectionmethod of TNF-α secretion are the same as described above, and will notrepeat them here. The detected result of TNF-α secretion was shown inFIG. 4A.

As shown in FIG. 4A, the active fractional interval (II) of HE-06 couldinhibit the secretion of TNF-α in LPS-induced acute inflammatory mice,and the inhibition level was about 16%, indicating that the activefractional interval (II) of HE-06 could in vivo inhibit TNF-α secretioninduced by inflammation.

Example 9: The Active Fractional Interval (II) of HE-06 can Improve theClinical Symptoms of EAE

Except for the drugs to be administered was replaced with the activefractional interval (II) of HE-06 and the administered active fractionalinterval (II) of HE-06 with the concentration of 0 mg/ml (as the controlgroup or vehicle), 30 mg/ml, 100 mg/ml and 300 mg/ml, the experimentalanimals, the established mouse autoimmune EAE model, and the scoringcriteria are the same as described above, and will not repeat them here.The experimental results were shown in FIG. 4B, and the group to beadministered with equal volume of solvent was considered as the controlgroup.

As shown in FIG. 4B, after induction of EAE, the mice have been appearedthe clinical symptoms of hind limb paralysis and severe forelimbweakness as above described Score 4 on 14^(th) day after onset, butadministration of the active fractional interval (II) of HE-06 with theconcentration of 30 mg/ml, 100 mg/ml and 300 mg/ml could slow down theprogress of EAE, and present a dose-dependent effect, indicating thatthe active fractional interval (II) of HE-06 can effectively improve theclinical symptoms in EAE animal model. In which * represents p value ofless than 0.05; ** represents p value of less than 0.01; *** representsp value of less than 0.001.

Example 10: The Active Fractional Interval (II) of HE-06 can Alleviatethe Symptom of Imiquimod (IMQ)-Induced Psoriasis-Like Dermatitis

(1) Experimental Animal

The 8 weeks BALB/c male mice were purchased from BioLASCO Taiwan Co.,Ltd. After acclimation and quarantine, the batch of mice wereapproximately 9-10 weeks of age at the time of performing the experimentof IMQ-induced psoriasis-like dermatitis. The experimental animals werebred in the environment: 12 hours light and 12 hours dark, roomtemperature of 23±2° C., and relative humidity of 40-70%. Duringfeeding, animals were free to obtain adequate food and drinking water.In addition, during the quarantine and testing period, the animals wereobserved and recorded by the veterinarian and the experimental staff ofITRI to ensure health of these experimental animals.

(2) Establishment of Mouse Model of Psoriasis-Like Dermatitis

Before the experiment, the mice were weighed and grouped so that theaverage body weight of each group was not significantly different. Then,the hair on the back of each mouse was shaved, and the shaving area wasabout 4*2 cm². Since day 0, 3.125 mg imiquimod (IMQ) was administered bysmearing to the skin of shaved area of each mouse daily for consecutive6 days. At the same time, the active fractional interval (II) of HE-06or solvent was also administered by performing tube feeding (10 ml/kg)or skin smearing (about 30˜50 mg/mouse) for 6 days, and these mice weresacrificed on the 6^(th) day with excess CO₂.

(3) Records and Data Analysis of the Degree of Skin Irritation

The above-mentioned degree of skin irritation is based on Psoriasis Areaand Severity Index (PASI), in which erythema, thickening, and scaling ofskin were assessed and analyzed for judging the scale. Scale 0-4 isdefined as follows: Scale 0 represents no obvious symptoms, Scale 1represents mild symptoms, Scale 2 represents moderate symptoms, Scale 3represents moderate to severe symptoms, and Scale 4 represents severesymptoms.

The mean value and standard error of mean (S.E.M.) of each group werecalculated by accumulating the index observed in the same mouse, andt-test statistics was used to distinguish the differences between thegroups. In which p value of less than 0.05 indicates that there exists astatistically significant difference between the two groups. Theexperimental results are shown in FIG. 4C.

As shown in FIG. 4C, after induction of psoriasis-like dermatitis byIMQ, the mice have been appeared the clinical symptoms of psoriasis-likedermatitis on 2^(nd) day after induction, such as erythematous skin,uneven thickness, and wrinkled dander, but administration of the activefractional interval (II) of HE-06 could alleviate the clinical symptomsof IMQ-induced psoriasis-like dermatitis. In which * represents p valueof less than 0.05; ** represents p value of less than 0.01.

Example 11: Isolation and Preparation of the Active Ingredient Compound,Vanillin or Tsaokoin (TK), of HE-06

The extract of A. tsao-ko, HE-06 was first extracted with a distillationstep by steam for 24 hours to remove the portion of essential oil. Then,a step of heating and refluxing with a low polarity solvent wasperformed for 8 hours. Thereafter, the extract was eluted by gradientelution with n-Hexane, EtOAc and MeOH, and the step of Silica Gel ColumnChromatography was further performed. Each fraction was analyzed andseparated by TLC and the active fraction was checked by performing theactivity test. Further, two compounds were isolated from the activefraction and determined to be vanillin and tsaokoin after analysis.Then, the fraction containing either vanillin or tsaokoin component wascombined respectively to obtain the active ingredient compound vanillinand tsaokoin of HE-06.

Example 12: The Active Ingredient Compound Tsaokoin (TK) can InhibitIL-17 and TNF-α Secretion

(1) IL-17 Secretion and MTT Test

Except for the drugs to be administered was replaced with activeingredient compound tsaokoin (TK) of HE-06, the utilized EL4 lymphomacells, the culture condition, the method of inflammation induction, andthe detection method of IL-17 secretion and MTT test are the same asdescribed above, and will not repeat them here. The detected results ofIL-17 secretion and MTT test were shown in FIG. 5A and Table 3.

TABLE 3 The inhibitory effect of the active ingredient compound TK onIL-17 secretion in EL4 murine lymphoma cells. IC₅₀ is the concentrationof TK causing 50% IL-17 secretion inhibition. Test sample IC₅₀ (μg/ml)Tsaokoin (TK) 12.1

The results of in vitro cell analysis shown in FIG. 5A indicated thatthe active ingredient compound TK of HE-06 can inhibit the secretion ofIL-17 in activated EL4 lymphoma cells.

(2) Test of Inhibition in TNF-α Secretion

Except for the drugs to be administered was replaced with the activeingredient compound tsaokoin (TK) of HE-06, the utilized BALB/c mice,the feeding condition, the mouse model of acute inflammation, and thedetection method of TNF-α secretion are the same as described above, andwill not repeat them here. The detected result of TNF-α secretion wasshown in FIG. 5B.

As shown in FIG. 5B, the active ingredient compound tsaokoin (TK) ofHE-06 could significantly inhibit the secretion of TNF-α in LPS-inducedacute inflammatory mice, indicating that TK could in vivo inhibit TNF-αsecretion induced by inflammation. In which *** represents p value ofless than 0.001.

Example 13: The Active Ingredient Compound Tsaokoin (TK) can Improve theClinical Symptoms of EAE

Except for the drugs to be administered was replaced with the activeingredient compound tsaokoin (TK) of HE-06 and the administered tsaokoinwith the concentration of 0 mg/ml (as the control group or vehicle), 30mg/ml, and 100 mg/ml, the experimental animals, the established mouseautoimmune EAE model, and the scoring criteria are the same as describedabove, and will not repeat them here. The experimental results wereshown in FIG. 5C, and the group to be administered with equal volume ofsolvent was considered as the control group.

As shown in FIG. 5C, after induction of EAE, the mice have been appearedthe clinical symptoms of hind limb paralysis and severe forelimbweakness as above described Score 4 on 14^(th) day after onset, butadministration of tsaokoin with the concentration of 30 mg/ml and 100mg/ml could slow down the progress of EAE, and present a dose-dependenteffect, indicating that tsaokoin can effectively improve the clinicalsymptoms in EAE animal model. In which * represents p value of less than0.05; ** represents p value of less than 0.01; *** represents p value ofless than 0.001.

Example 14: The Active Ingredient Compound Tsaokoin (TK) can Alleviatethe Symptom of Imiquimod (IMQ)-Induced Psoriasis-Like Dermatitis

Except for the drugs to be administered was replaced with the activeingredient compound tsaokoin (TK) of HE-06 and the administered tsaokoinwith the concentration of 100 mg/ml, the experimental animals, theestablished model of psoriasis-like dermatitis, and the scoring criteriaare the same as described above, and will not repeat them here. Theexperimental results were shown in FIG. 5D, and the group to beadministered with equal volume of solvent was considered as the controlgroup.

As shown in FIG. 5D, after induction of psoriasis-like dermatitis byIMQ, the mice have been appeared the clinical symptoms of psoriasis-likedermatitis on 2^(nd) day after induction, such as erythematous skin,uneven thickness, and wrinkled dander, but administration of tsaokoincould significantly alleviate the clinical symptoms of IMQ-inducedpsoriasis-like dermatitis. In which * represents p value of less than0.05; ** represents p value of less than 0.01.

It will be apparent to those skilled in the art that variousmodifications and variations can be made to the disclosed embodiments.It is intended that the specification and examples be considered asexemplary only, with a true scope of the disclosure being indicated bythe following claims and their equivalents.

What is claimed is:
 1. A pharmaceutical composition for treating oralleviating autoimmune-related diseases, comprising: an extract ofAmomum tsao-ko as an effective ingredient and a pharmaceuticallyacceptable carrier or salt.
 2. The pharmaceutical composition as claimedin claim 1, wherein the extract of Amomum tsao-ko is extracted from aroot, a stem, a leaf, a flower, a fruit, a seed, or a combinationthereof.
 3. The pharmaceutical composition as claimed in claim 1,wherein the extract of Amomum tsao-ko is obtained from performing anextraction with an organic solvent.
 4. The pharmaceutical composition asclaimed in claim 3, wherein the organic solvent comprises alcohol,ester, alkane, haloalkane solvent or a combination thereof.
 5. Thepharmaceutical composition as claimed in claim 4, wherein the organicsolvent is 50-95% alcohol solvent.
 6. The pharmaceutical composition asclaimed in claim 3, wherein a volume of the organic solvent is 3-10times the weight of Amomum tsao-ko.
 7. The pharmaceutical composition asclaimed in claim 3, wherein the extraction of the extract of Amomumtsao-ko is performed with an extraction temperature of 10 to 35° C. andan extraction time of 3 to 10 days.
 8. The pharmaceutical composition asclaimed in claim 3, wherein the extraction of the extract of Amomumtsao-ko is further performed with a shaking mode.
 9. The pharmaceuticalcomposition as claimed in claim 8, wherein the extraction of the extractof Amomum tsao-ko further comprises a distillation step.
 10. Thepharmaceutical composition as claimed in claim 9, wherein the extractionof the extract of Amomum tsao-ko further comprises a heating reflux stepwith a low polarity solvent.
 11. The pharmaceutical composition asclaimed in claim 1, wherein the pharmaceutical composition has theeffect on inhibiting cytokine secretion, wherein the cytokine includesIL-17, TNF-α, and a combination thereof.
 12. The pharmaceuticalcomposition as claimed in claim 1, wherein the autoimmune-relateddiseases is selected from the group consisting of Multiple Sclerosis(MS), neuromyelitis optica (NMO), acute disseminated encephalomyelitis(ADEM), demyelination-related neuritis, Ankylosing spondylitis,Psoriasis, Psoriatic arthritis, Rheumatoid arthritis (RA), Crohn'sdisease, Juvenile idiopathic arthritis (JIA), and Ulcerative Colitis(UC).
 13. The pharmaceutical composition as claimed in claim 1, whereinthe autoimmune-related diseases is Multiple Sclerosis (MS).
 14. Thepharmaceutical composition as claimed in claim 1, wherein thepharmaceutical composition is administered orally, non-orally,parenterally by an inhalation spray, or via an implanted reservoir. 15.A method of treating or alleviating autoimmune-related diseases,comprising: administering a pharmaceutical composition to a subject inneed thereof, wherein the pharmaceutical composition comprises anextract of Amomum tsao-ko as an effective ingredient and apharmaceutically acceptable carrier or salt.
 16. A pharmaceuticalcomposition for treating or alleviating autoimmune-related diseases,comprising: a compound as an effective ingredient and a pharmaceuticallyacceptable carrier or salt, wherein the compound is vanillin, tsaokoin,or a combination thereof.
 17. The pharmaceutical composition as claimedin claim 16, wherein the pharmaceutical composition has the effect oninhibiting cytokine secretion, wherein the cytokine includes IL-17,TNF-α, and a combination thereof.
 18. The pharmaceutical composition asclaimed in claim 16, wherein the autoimmune-related diseases is selectedfrom the group consisting of Multiple Sclerosis (MS), neuromyelitisoptica (NMO), acute disseminated encephalomyelitis (ADEM),demyelination-related neuritis, Ankylosing spondylitis, Psoriasis,Psoriatic arthritis, Rheumatoid arthritis (RA), Crohn's disease,Juvenile idiopathic arthritis (JIA), and Ulcerative Colitis (UC). 19.The pharmaceutical composition as claimed in claim 16, wherein theautoimmune-related diseases is Multiple Sclerosis (MS).
 20. Thepharmaceutical composition as claimed in claim 16, wherein thepharmaceutical composition is administered orally, non-orally,parenterally by an inhalation spray, or via an implanted reservoir. 21.A method of treating or alleviating autoimmune-related diseases,comprising: administering a pharmaceutical composition to a subject inneed thereof, wherein the pharmaceutical composition comprises acompound as an effective ingredient and a pharmaceutically acceptablecarrier or salt, wherein the compound is vanillin, tsaokoin, or acombination thereof.